• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.102-102
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• 2003

Embryonic stem (ES) cells proliferate extensively in the undifferentiated state and have the potential to differentiate into a variety of cell types in response to various environmental cues. The generation of functional dopaminergic neurons from ES cells is promising for cell replacement therapy to treat Parkinson's disease. We compared the in vitro differentiation potential of pluripotent human embryonic stem (hES, MB03) cells induced with basic fibroblast growth factor (bFGF) or retinoic acid (RA). Both types of treatment resulted in similar neural cell differentiation patterns at the terminal differentiation stage, specifically, 75% neurons and 11% glial cells. Additionally, treatment of hES cells with brain derived neurotrophic factor (BDNF) or transforming growth factor (TGF)- $\alpha$ during the terminal differentiation stage led to significantly increased tyrosine hydroxylase (TH) expression, compared to control (P<0.05). In contrast, no effect was observed on the rate of mature or glutamic acid decarboxylase-positive neurons. Immunostaining and HPLC analyses revealed the higher levels of TH (20.3%) and dopamine in bFGF and TGF-$\alpha$ treated hES cells than in RA or BDNF treated hES cells. The results indicate that TGF-$\alpha$ may be successfully used in the bFGF induction protocol to yield higher numbers of functional dopaminergic neurons from hES cells.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.19-19
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• 2002

This study was to examine whether the in vitro differentiated neural cells derived from human embryonic stem (hES, MB03) cells can be survived and expressed tyrosin hydroxylase(TH) in grafted normal or PD rat brain. To differentiate in vitro into neural cells, embryoid bodies (EB: for 5 days, without mitogen) were formed from hES cells, neural progenitor cells(neurosphere, for 7-10 days, 20 ng/㎖ of bFGF added N2 medium) were produced from EB, and then finally neurospheres were differentiated into mature neuron cells in N2 medium(without bFGF) for 2 weeks. In normal rat brain, neural progenitor cells or mature neuron cells (1×10/sup 7/ cells/㎖) were grafted to the striatum of normal rats. After 2 weeks, when the survival of grafted hES cells was examined by immunohistochemical analysis, the neural progenitor cell group indicated higher BrdU, NeuN＋, MAP2＋ and GFAP＋ than mature neuron cell group in grafted sites of normal rats. This result demonstrated that the in vivo differentiation of grafted hES cells be increased simultaneously in both of neuronal and glial cell type. Also, neural progenitor cell grafted normal rats expressed more TH pattern than mature neuron cells. Based on this data, as a preliminary test, when the neural progenitor cells were grafted into the striatum of 6-hydroxydopamine lesioned PD rats, we confirmed the cell survival (by double staining of Nissl and NeuN) and TH expression. This result suggested that in vitro differentiated neural progenitor cells derived from hES cells are more usable than mature neuron cells for the neural cell grafting in animal model and those grafted cells were survived and expressed TH in normal or PD rat brain.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.95-95
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• 2002

Human embryonic stem (hES) cells can be induced to differentiate into tyrosine hydroxylase expressing (TH+) cells that may serve as an alternative for cell replacement therapy for Parkinson's disease (PD). To examine in vitro differentiation of hES (MB03, registered in NIH) cells into TH+ cells, hES cells were induced to differentiate according to the 4-/4+ protocol using retinoic acid (RA), ascorbic acid (AA), and/or lithium chloride (LiCl) followed by culture in N2 medium for 14 days, during which time the differentiation occurs. Immunocytochemical stainings of the cells revealed that approximately 21.1% of cells treated with RA plus AA expressed TH protein that is higher than the ratio of TH+ cells seen in any other treatment groups (RA, RA+LiCl or RA+AA+LiCl). In order to see the differentiation pattern in vivo and the ability of in vitro differentiation-induced cells in easing symptomatic motor function of PD animal model, cells (2 $\times$ 10$^{5}$ cells/2${mu}ell$) undergone 4-/4+ protocol using RA plus AA without any further treatment were transplanted into unilateral striatum of MPTP-lesioned PD animal model (C57BL/6). Following the surgery, motor behavior of the animals was examined by measuring the retention time on an accelerating rotar-rod far next 10 weeks. No significant differences in retention time of the animals were noticed until 2 weeks post-graft; however, it increased markedly at 6 weeks and 10 weeks time point after the surgery. Immunohistochemical studies confirmed that a reasonable number of TH+ cells were found at the graft site as well as other remote sites, showing the migrating nature of embryonic stem cells. These results suggest that in viかo differentiated hES cells relieve symptomatic motor behavior of PD animal model and should be considered as a promising alternative for the treatment of PD.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.273-273
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• 2004

This study was to examine in vitro neural cell differentiation pattern of the genetically modified human embryonic stem cells expressing tyrosine hydroxylase (TH). Human embryonic stem (hES, MB03) cell was transfected with cDNAs cording for TH. Successful transfection was confirmed by western immunoblotting. (omitted)

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.103-103
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• 2003

The purpose of this study is to evaluate an efficacy of in vitro differentiated human embryonic stem (hES, MB03) cells expressing Nurr1 in relief of symptomatic motor behavior of Parkinson's disease (PD) animal models MB03 was genetically modified to express Nurr1 protein and was induced to differentiate according to 2-/4+ protocol using retinoic acid and ascorbic acid. The differentiation-induced cells were selected for 10 to 20 days thereafter in N2 medium. Upon selection, cells expressing GFAP, TH, or NF200 were 38.8%, 11%, and 20.5%, respectively. in order to examine therapeutic effects of the differentiated cells in PD animal model, rats were unilaterally lesioned by administration of 6-kydroxydopamine HCI (6-OHDA) into medial forebrain region (MFB, AP -4.4 mm, ML 1.2 mm, DV 78 mm with incision bar set at -2.4 mm), as a reference to bregma and the surface of the skull. Confirmation of successful lesion by apomorphine-induced rotational behavior, differentiated cells were transplanted into the striatum (AP 1.0, ML 3.5, DV -5.0; AP 0.6, ML 2.5, DV -4.5). Improvements of asymmetric motor behavior by the transplantation were examined every two weeks after the surgery. In two weeks, numbers of rotation by the experimental rats were $-14.8 \pm 33.9%$ (P<0.05) of the number before transplantation, however, the ratio increased slightly to $13.6 \pm 56.3%$ in six weeks. In contrast, the ratio of sham-grafted animals ranged from 112.3+8.5% to 139.2+28.9% during the examination. Immunohistochemical studies further confirmed the presence, survival, migration, and expression of TH of the transplanted human cells.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.117-127
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• 2002

Objective: This study was to investigate the generation of the functional neuron derived from human embryonic stem (hES, MB03) cells on in vitro neural cell differentiation system. Methods: For neural progenitor cell formation derived from hES cells, we produced embryoid bodies (EB: for 5 days, without mitogen) from hES cells and then neurospheres (for $7{\sim}10$ days, 20 ng/ml of bFGF added N2 medium) from EB. And then finally for the differentiation into mature neuron, neural progenitor cells were cultured in i) N2 medium only (without bFGF), ii) N2 supplemented with 20 ng/ml platelet derived growth factor-bb (PDGF-bb) or iii) N2 supplemented with 5 ng/ml brain derived neurotrophic factor (BDNF) for 2 weeks. Identification of neural cell differentiation was carried out by immunocytochemistry using $\beta_{III}$-tubulin (1:250), MAP-2 (1:100) and GFAP (1:500). Also, generation of functional neuron was identified using anti-glutamate (Sigma, 1:1000), anti-GABA (Sigma, 1:1000), anti-serotonin (Sigma, 1:1000) and anti-tyrosine hydroxylase (Sigma, 1:1000). Results: In vitro neural cell differentiation, neurotrophic factors (PDGF and BDNF) treated cell groups were high expressed MAP-2 and GFAP than non-treated cell group. The highest expression pattern of MAP-2 and $\beta_{III}$-tubulin was indicated in BDNF treated group. Also, in the presence of PDGF-bb or BDNF, most of the neural cells derived from hES cells were differentiated into glutamate and GABA neuron in vitro. Furthermore, we confirmed that there were a few serotonin and tyrosine hydroxylase positive neuron in the same culture environment. Conclusion: This results suggested that the generation of functional neuron derived from hES cells was increased by addition of neurotrophic factors such as PDGF-bb or BDNF in b-FGF induced neural cell differentiation system and especially glutamate and GABA neurons were mainly produced in the system.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.101-101
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• 2003

Main strategy for a treatment of Parkinson's disease (PD), due to a progressive degeneration of dopaminergic neurons, is a pharmaceutical supplement of dopamine derivatives or ceil replacement therapy. Both of these protocols have pros and cons; former exhibiting a dramatic relief but causing a severe side effects on long-term prescription and latter also having a proven effectiveness but having availability and ethical problems Embryonic stem (ES) cells have several characteristics suitable for this purpose. To investigate a possibility of using ES cells as a carrier of therapeutic gene(s), human ES (hES, MB03) cells were transfected with cDNAs coding for tyrosine hydroxylase (TH) in pcDNA3.1 (+) and the transfectants were selected using neomycin (250 $\mu /ml$). Expression of TH being confirmed, two of the positive clone (MBTH2 & 8) were second transfected with GTP cyclohydrolase 1 (GTPCH 1) in pcDNA3.1 (+)-hyg followed by selection with hygromycin-B (150 $\mu /ml$) and RT-PCR confirmation. By immune-cytochemistry, these genetically modified but undifferentiated dual drug-resistant cells were found to express few of the neuronal markers, such as NF200, $\beta$-tubulin, and MAP2 as well as astroglial marker GFAP. This results suggest that over-production of BH4 by ectopically expressed GTPCH I may be involved in the induction of those markers. Transplantation of the cells into striatum of 6-OHDA- denervated PD animal model relieved symptomatic rotational behaviors of the animals. Immunohistochemical analyses showed the presence of human cells within the striatum of the recipients. These results suggest a possibility of using hES cells as a carrier of therapeutic gene(s).